PubMed Health⌕ Search

Biomedical subjects

S Isojima

Publications and source records attributed to S Isojima.

At least 19 recordsLinked to original sources
PubMed1992

Antifertility effect of active immunization with ZP4 glycoprotein family of porcine zona pellucida in hamsters.

Female golden hamsters were immunized with solubilized porcine zona pellucida (s-PZP) or ZP4 glycoprotein family isolated from s-PZP by preparative SDS-PAGE. Both antigen preparations induced production of antibodies which reacted not only with porcine zona pellucida but also with the hamster zona pellucida. The hamsters immunized with solubilized porcine zona pellucida mainly produced antibodies reactive to ZP3, while the hamsters immunized with ZP4 mainly produced antibodies reactive to ZP4. The former animals became permanently infertile but the infertility in the latter animals was temporary and they became pregnant later. Histological studies revealed that the ovarian follicles in hamsters immunized with s-PZP were completely destroyed leaving only atrophic follicle-like cell clusters, while in the ovaries of hamsters immunized with ZP4 a number of small follicles with oocytes remained intact. These observations are encouraging for the further characterization of the ZP4 antigens as candidates for the development of a contraceptive vaccine.

Animals↗
PubMed1992

Sperm immobilizing antibodies react to the 3-O-sulfated galactose residue of seminolipid on human sperm.

It is well known that very few women who possess sperm immobilizing antibodies in their sera can conceive naturally even though there are no abnormalities in their reproductive organs on routine medical examination. A monoclonal antibody (MAb), designated 2H12, was produced by immunizing a BALB/c mouse with the human choriocarcinoma cell line JEG-3. MAb 2H12 showed strong sperm immobilizing activities and reacted to sulfatide and seminolipids. The sperm immobilizing activities of 2H12 were clearly absorbed with sulfatide or seminolipid whilst several other sperm immobilizing MAbs that were made by immunization with human sperm or seminal plasma could not be absorbed with the same sulfoglycolipids. The sperm immobilizing antibodies in the sera of infertile women with unknown cause were also clearly absorbed with sulfatide or seminolipid. MAb 2H12-conjugated immunobeads (MAb 2H12-IMBs) bound to motile sperm. This binding of 2H12-IMBs to sperm was competitively inhibited either by 2H12 or women's sera containing sperm immobilizing antibodies, but not by normal women's sera or several other sperm immobilizing MAbs to human sperm. These results suggest that the sperm immobilizing antibody in women's sera is directed against the 3-O-sulfogalactose residue of seminolipid on the sperm membrane.

Animals↗
PubMed1992

Comparative studies of the antigens recognized by sperm-immobilizing monoclonal antibodies.

Characteristic properties of the antigens recognized by sperm-immobilizing monoclonal antibodies (SI-mAbs) from different sources were compared by ELISA competitive inhibition assay, Western blot analysis, chromatographic analysis, and enzymatic digestion studies. Among 9 SI-mAbs, human mAb H6-3C4 and three mouse mAbs--2C6, 2B6, and 2E5--also possessed strong sperm-agglutinating activity. Binding of human mAb H6-3C4 to sperm was strongly inhibited by the three mouse mAbs (2C6, 2B6, and 2E5), but not by the rat or the other four mouse mAbs. SDS-PAGE revealed that mAb H6-3C4 and three mouse mAbs recognized the same antigen molecules of 15-25 kDa present in both sperm extracts and seminal plasma. Chemical treatments with trifluoromethanesulfonic acid and sodium metaperiodate destroyed the antigen determinants recognized by the above four mAbs, as detected by both ELISA and antibody absorption tests. Western blot analysis revealed that the antigens were susceptible to treatments with papain, proteinase K, and N-glycanase, but resistant to trypsin, V8 protease, and thermolysin. These results indicate that one of the major antigens recognized by mAbs with sperm-immobilizing action may be a sperm membrane-associated glycoprotein of 15-25 kDa and the epitope may involve N-linked oligosaccharides.

Amidohydrolases↗
PubMed1991

Further characterization of the porcine zona pellucida antigen corresponding to monoclonal antibody 3A4-2G1 exclusively cross-reactive with porcine and human zonae pellucidae.

Biochemical and molecular analyses were performed for the porcine zona pellucida (ZP) antigen corresponding to the monoclonal antibody (Mab) 3A4-2G1 exclusively cross-reactive with porcine and human zonae pellucidae (ZPe). By SDS-PAGE and Western blot analysis, Mab 3A4-2G1 reacted with a 92-kDa component of solubilized porcine ZP (s-PZP) under non-reducing conditions, while reacting with a 23-kDa component under reducing conditions. Diagonal SDS-PAGE revealed that the 92-kDa component was composed of 69 kDa and 23 kDa molecules. In two-dimensional O'Farrell's electrophoresis, the antigen molecules reactive to Mab 3A4-2G1 were distributed over a wide range of PI as a family of proteins around 92 kDa and 23 kDa regions under non-reducing and reducing conditions, respectively. A murine antiserum raised to the isolated 23-kDa component reacted not only with the 23-kDa but also with the 92-kDa glycoprotein family. The anti-23 kDa serum showed a strong inhibitory effect on human sperm-egg interaction by in vitro fertilization. The reactivity of s-PZP to Mab 3A4-2G1 was markedly reduced by treatments with proteolytic enzymes but not significantly with glycosidic enzymes. However, chemical deglycosylation of the antigen markedly reduced the antigen's activity. These results suggested that Mab 3A4-2G1 recognized a conformational antigen epitope structure of ZP.

Animals↗
PubMed1991

Sperm immobilizing and fertilization-blocking monoclonal antibody 2C6 to human seminal plasma antigen and characterization of the antigen epitope corresponding to the monoclonal antibody.

A monoclonal antibody (Mab 2C6) with strong sperm immobilizing and agglutinating activities was generated by cell fusion between spleen cells from a mouse immunized with human seminal plasma (HSP) and mouse myeloma cells. It also showed a strong inhibitory effect on human sperm-egg interaction. The corresponding antigen was present on the whole surface of ejaculated spermatozoa. In male genital organs, immunostaining with Mab 2C6 was observed in epididymis and seminal vesicle but not in testis. By Western blotting, immunostaining with Mab 2C6 was detected around the 15-25 kDa region under both reducing and non-reducing conditions. The antigen corresponding to Mab 2C6 was susceptible to treatment with periodate or trifluoromethanesulfonic acid. The antigenic activities were slightly increased by treatment with neuraminidase but reduced by further treatment with glycosidases. Enzymatic digestions with pronase and papain also reduced the antigenic activities. The antigen molecules exhibited a strong binding affinity to RCA lectin. These results indicated that Mab 2C6 recognized one of the components which might be secreted from epididymis or seminal vesicle and bind to ejaculated spermatozoa as a sperm coating antigen. The corresponding antigen seems to be a glycoprotein and its carbohydrate moiety has an important role in the conformation of the antigen epitope.

Animals↗
PubMed1991

Recognition of carbohydrate antigen epitopes by sperm-immobilizing antibodies in sera of infertile women.

STUDY OBJECTIVE: To study carbohydrate natures in the antigen epitopes corresponding to sperm-immobilizing antibodies in infertile women. DESIGN: Antibody absorption with human sperm and seminal plasma before and after treatments with trifluoromethanesulfonic acid or sodium metaperiodate. PATIENTS: Thirty-three patients who showed a positive sperm immobilization test provided their sera for the experiment. RESULTS: In 25 patients' sera whose sperm-immobilizing antibodies were absorbed with human seminal plasma, the antibody absorbing capabilities were completely abolished by deglycosylation treatment with trifluoromethanesulfonic acid. The sperm-immobilizing antibodies in 4 patients' sera were absorbed out with sperm membrane fraction before the treatment but not after the treatment with trifluoromethanesulfonic acid. In some patients' sera, the antibody-absorbing capabilities of ejaculated sperm were markedly reduced by sodium metaperiodate treatment. CONCLUSION: The majority of sperm-immobilizing antibodies in infertile patients might be generated to carbohydrate structures of the sperm-coating antigens or sperm membrane antigens.

Antibodies↗
PubMed1991

Blocking of human sperm-zona interaction by monoclonal antibodies to a glycoprotein family (ZP4) of porcine zona pellucida.

To study zona pellucida antigens involved in human fertilization, five monoclonal antibodies (MAbs)--2A1, 2G3, 4A2, 4E12, and 5H4--were produced to a glycoprotein family (ZP4) isolated from heat-solubilized porcine zonae pellucidae. Each MAb reacted not only with solubilized porcine zona glycoproteins but also with the glycoproteins deglycosylated by trifluoromethanesulfonic acid treatment. They also reacted with intact zonae pellucidae of porcine and human oocytes. Three (4A2, 4E12, and 5H4) of the five MAbs showed a significant blocking effect on human sperm binding and penetration of human zonae pellucidae. The 5H4 MAb showed a strong reaction with ZP4 and ZP1 glycoprotein families of porcine zonae pellucidae, and four other MAbs reacted more strongly with ZP3 than with ZP4. The reactivity of 5H4 with porcine zona glycoproteins was destroyed by chymotrypsin digestion, but the antigen epitope was resistant to proteolysis by trypsin and endoproteinase Lys-C. A peptide fragment reactive to 5H4 was isolated by reverse-phase HPLC from endoproteinase Lys-C-treated ZP4 glycoproteins, and its molecular mass was determined to be 7 kDa by SDS-PAGE. These results suggested that the antigen epitope corresponding to 5H4 is a good candidate for development of a contraceptive vaccine.

Animals↗
PubMed1991

Isolation of four major glycoprotein families (ZP1, ZP2, ZP3, ZP4) of porcine zona pellucida and characterization of antisera raised to each glycoprotein family.

Porcine zona pellucida (ZP) contains antigens which cross-react with man, and anti porcine ZP antibody was found to exhibit a strong blocking effect on human sperm-ZP interaction. Previously, we found that porcine ZP was composed of four major glycoprotein families (ZP1, ZP2, ZP3, ZP4). In this study, each of the glycoprotein families was isolated by O'Farrell's two-dimensional electrophoresis and injected into mice to obtain antisera to each component. All the mouse antisera had high titers of antibodies to porcine and human ZP in an immunofluorescent staining test. Western blot analysis showed that although the antisera most strongly reacted with the corresponding components of porcine ZP, each glycoprotein family also possessed antigens which cross-reacted with each other. The antisera to ZP1, ZP3 and ZP4 strongly inhibited human sperm-ZP interaction but antiserum to ZP2 had no inhibitory effect on it.

Animals↗
PubMed1990

Efficacy of the high molecular weight fraction of plasma for the maintenance of pregnancy associated with thrombotic thrombocytopenic purpura.

We have investigated the methods for the maintenance of a pregnancy in a patient with thrombotic thrombocytopenic purpura (TTP), said condition, since 1984, having been controlled by a plasma infusion every 3 to 4 weeks. In a preliminary trial it was confirmed that an infusion of the high molecular weight fraction (HMW-F) of plasma, separated by an Evaflux 2A fractionator, improved the patient's thrombocytopenia as the plasma infusion, and maintained its beneficial effect for about 2 weeks during early pregnancy. Though an occurrence of a toxemia-like syndrome responded to repeated plasma infusion, the dose of plasma required to improve the thrombocytopenia gradually increased and reached 5,040 ml by the 20th week of pregnancy. Thus, instead of periodic infusions of whole plasma, periodic infusions of the HMW-F of plasma were used. Under this regimen the platelet count remained above 10.0 x 10(4)/microliters during late pregnancy, and the total dose (2,600 ml) of HMW-F of plasma that was administered until delivery at full term was less than the dosage of whole plasma that was used during early pregnancy. In this manner we were able to obtain a healthy baby by controlling the patient's TTP during pregnancy. This method of preventing thrombocytopenia appears to be safer with respect to volume loading during pregnancy in the TTP patient.

Adult↗
PubMed1990

Monoclonal antibody recognizing an apparent peptide epitope of human seminal plasma glycoprotein and exhibiting sperm immobilizing activity.

A hybridoma (3B2-F7) has been established which secretes a monoclonal antibody (Mab) directed against a peptide determinant of human seminal plasma glycoprotein (HSP-gP). The deglycosylation of HSP-gP was performed chemically with TFMS hydrolysis and enzymatically in the presence of detergent and further treated with periodic acid after fixing deglycosylated HSP on plastic wells. The Mab 3B2-F7 (IgM, kappa) exhibited sperm immobilization activity (256 units of SI50) and inhibited sperm binding to human zona pellucida. Human epididymis, pancreatic islets of Langerhan's and distal tubulus of kidney were strongly labelled whilst other tissues were essentially negative by avidin-biotin complex tissue staining with this Mab. The antigen epitope to the Mab was in the 36 kDa molecule of human HSP-gP. The antigenic determinant recognized by Mab 3B2-F7 was destroyed by six different proteases, but was resistant to N-glycanase and other carbohydrate splitting enzymes. This epitope is therefore likely to be composed of a polypeptide chain. Peptide fragments after proteolysis of the HSP molecule with Staph. aureus V8 protease and trypsin retained antigenicity, hence the epitope corresponding to the Mab may be a peptide chain and not dependent on the conformational structure of the polypeptide.

Animals↗
PubMed1990

Correlation between quantitative antibody titers of sperm immobilizing antibodies and pregnancy rates by treatments.

Immunological infertility in women who possessed sperm immobilizing (SI) antibodies made it very difficult to conceive using the usual treatments. We examined SI antibodies by the quantitative Sperm Immobilization Test and found the antibody titers (50% sperm immobilization unit: SI50 unit) associated with pregnancy rates. Patients with high SI50 titers (greater than 10 units) did not conceive by ordinary or repeated artificial inseminations with husband's semen (AIH) except when treated with in vitro fertilization (IVF) and embryo replacement. Patients with relatively low SI50 titers (less than 10 units) could conceive either by repeated or ordinary AIH, though the success rates were lower than by IVF-embryo replacement. It is important to assess the SI50 titers by the quantitative method to select treatments for infertile women with SI antibodies. In follow-up studies of the patients who conceived successfully, it was found that SI50 titers tended to decline as pregnancy proceeded.

Antibodies↗
PubMed1990

[Reverse passive hemagglutination assay for human chorionic gonadotropin in urine using monoclonal antibody].

Aiming to find a urinary hCG immuno-assay which is specific, sensitive and easy to perform, a reverse passive hemagglutination reaction was studied by using sheep red blood cells (SRBC) coupled with monoclonal antibodies (Mab) to hCG. Three Mabs (5D4, 6E4, 2F8) with different specialty were used for the study. Mab 5D4 reacted to hCG, hCG-beta, and LH but not to hCG-alpha. Mab 6E4 reacted to hCG, hCG-alpha and LH, but not to hCG-beta. Mab 2F8 reacted to hCG but not to hCG-alpha, hCG-beta, or LH. All three Mabs were IgG1. SRBC were treated with glutaraldehyde and then with tannic acid. These treated SRBC were coupled with IgG(2mg/ml) of each anti hCG-Mab. For assays, 30 microliters of 1:1 mixtures of two different Mab-coupled SRBC and 30 microliters of standard hCG or urine samples were mixed in wells of microtiter plates and reacted for 60 min at room temperature. Among three different combinations, the couple 5D4-SRBC and 2F8-SRBC were most sensitive and specific for hCG assays and the minimum amount of hCG and LH detected in this combination assays was 12.5 mIU/ml and 800 mIU/ml, respectively. Some clinical data obtained by applying this assay were presented.

Antibodies, Monoclonal↗
PubMed1989

Characterization of epitopes of seminal plasma antigen stimulating human monoclonal sperm-immobilizing antibodies: a personal review.

In observations made between 1974 and 1984, 40 of 303 women with unexplained sterility (13.2%) showed positive sperm-immobilizing antibodies. Among many kinds of antibodies to human sperm including sperm coating antigens, the biologically active antibodies, such as sperm-immobilizing agglutinating antibodies and blocking antibodies for fertilization, could be relevant to infertility. Harmless sperm-binding antibodies are present even in the sera and cervical mucus of fertile women. Antigens corresponding to monoclonal antibodies (1C4, 2C6, 2E5), which were generated to human sperm coating antigens and indicated strong sperm-immobilizing activities, seemed to have carbohydrate epitopes. The majority of women with sterility of unknown cause appeared to raise sperm-immobilizing antibodies to carbohydrate epitopes of sperm. The stable human-mouse heterohybridoma H6-3C4 secreting monoclonal antibody (IgM, lambda) with extremely high titres of sperm-immobilizing (SI50, 5000 units) and agglutinating (1:1600) activities was successfully established from peripheral lymphocytes of a sterile woman. The chemical structure of an antigen epitope corresponding to human monoclonal antibody H6-3C4 was found to consist of internally repetitive, unbranched N-acetyllactosamine (blood type i antigen). Ejaculated human sperm appeared to be densely covered with sialyl blood type i antigen and sialyl branched internally repetitive N-acetyllactosamine (sialyl blood type I antigen). The antibody-producing V-H and V-L genes of the human hybridoma H6-3C1 were cloned and preserved to stabilize antibody production. Class-switch variants of heavy chain from mu to gamma (IgG1, lambda) in human Mab H6-3C4 were produced by recombinant DNA technology.

Antibodies, Monoclonal↗
PubMed1989

Techniques for sperm immobilization test.

Several semiquantitative, quantitative, and microassay techniques had been developed to detect antibodies bound to human spermatozoa: sperm agglutination test (SAT), sperm immobilization test (SIT), immunofluorescence test, radioantiglobulin test, enzyme-linked immunosorbent assay (ELISA), mixed erythrocyte-spermatozoa antiglobulin reaction (MAR), "Panning" test, and immunobead test (IBT). Clinical application of these techniques include (a) detection of sperm immobilizing antibodies in sera of sterile women, (b) follow-up study of sperm immobilizing antibodies, and (c) detection of sperm immobilizing antibodies in cervical mucus and other secretions. The chemical structure of antigen epitope corresponding to Mab H6-3C4 may recognize the internally located repetitive unbranched N-acetyllactosamine structure, regardless of terminal substitution at Gal (i.e., sialyl-i as well as i structure). The majority of sperm-immobilization (SI) positive women's sera were absorbed with carbohydrate components on ejaculated sperm, but only one serum competed with Mab H6-3C4 on binding to sperm except a serum from whom lymphocytes were donated to make Mab H6-3C4. The SI agglutinating antibodies (Abs) in women's sera were raised to the carbohydrate epitopes of glycoprotein in HSP, but epitopes might have several different conformational structures. Studies are in progress to find whether or not SI-Abs could be generated to peptide epitope of human seminal plasma (HSP) or sperm.

Agglutination Tests↗
PubMed1989

[Role of sperm passage through cervical mucus: fertilizing capacity tested by in vitro fertilization with zona-free hamster eggs].

The effect of sperm passage through cervical mucus (CM) on the fertilizing capacity of human spermatozoa was examined in the in vitro fertilization system of zona-free hamster eggs. Each drop of ejaculated semen and BWW culture medium was connected by a small capillary tube filled with preovulatory CM, egg white or BWW medium under liquid paraffin oil in a plastic petri dish. After 2 hours, zona-free hamster eggs were added to the drop of BWW culture medium containing spermatozoa which had passed through the capillary tube and the mixture was incubated for various lengths of time at 37 degrees C under 5% CO2 in air. Human spermatozoa, which were washed and preincubated for 2 hours in BWW medium, were capable of fertilizing zona-free hamster eggs but needed a longer incubation time than spermatozoa which had passed through CM. Fertilization rates of spermatozoa which had passed through CM and egg white were very similar, but no fertilization occurred in the drop containing spermatozoa which had passed through BWW medium, presumably because of the contamination with seminal plasma. These results indicate that the most important role of CM may be to separate motile spermatozoa from seminal plasma components hostile to fertilization.

Animals↗